This invention relates to a method and apparatus for quantatively determining a degree of agglutination of particles with the help of optical means and more particularly is concerned with the optical measurement of the state of agglutination of particles while making them agglutinate through the antigen-antibody reaction by using sensitized particles being coated with an emulsion containing either antigen or antibody.
The cognition of the state or existence of the agglutination in a suspension of particles is helpful to grasp the chemical or mechanical properties, stability, and reactivity of it.
In particular, the agglutination reaction--in which sensitized particles such as latex, bentonite, or kaolin being coated with an emulsion containing either antigen or antibody are made to react with the other inversely corresponding antibody or antigen being possibly contained in a liquid to be tested--is being put to practical use as a simple and easy measuring method of immunochemical ingredients. Best of all, the agglutination reaction carried on in latex composed of polystyrene and the like is serviceable especially at the time of measuring various sorts of proteins and hormones including RF (rheumatoid factor), CRP (c-reactive protein), and others because of the detecting sensitivity of high grade with an excellent specificity.
For the time being, however, the measurement of the agglutination reaction of such a kind of latex is made as a rule depending on the decision of the presence of the latex which has agglutinated through the antigen-antibody reaction in making the sensitized latex liquid react with the liquid to be tested usually on slide glass or plate, on the basis of eye measurement, otherwise on some occasion by the use of optical means. The results of measurement in both cases mentioned above may be stated as having done throughout in a qualitative way. Especially, in the case of eye measurement, it is ready to cause each time the obscurity in deciding the presence of the agglutination.
Further, in the case of resorting to the optical measuring method, and aiming at the quantitative measurement at that, it is necessary to prepare a series of test objects having been diluted in stages, observe the presence of the agglutination about each sample out of the series while manipulating the reaction, and take a measure to indicate the quantity of antigen or antibody by detecting which stage of dilution forms the boundary layer discriminating the state of agglutination from the state of non-agglutination. Such being the case, the procedure as mentioned above has such a marked imperfection that needs much labor and time or a great deal of latex reagent at the time of measuring even one sample.
On the other hand, there is a way of measuring the light transmissivity of the liquid in the cell by means of a light of wave length belonging to the near infra-red region with the object of quantatively determining a degree of agglutination. In this case, quite a hard point to settle lies in that inasmuch as the clots of agglutinated latex and the other particles of non-agglutinated latex coexist within the observation area of the example, the measurement of the transmissivity, for example, cannot help being done on the mixed system of the clots of agglutinated latex and the particles of non-agglutinated latex, as a result bringing about the drop in its sensitivity. It makes no difference even if some scattered light would be used. This method has another defect that the reproducibity of the measurement done is not good because the agglutinated clots are scattering distributed.
Further, there is also a method for quantitatively determining a degree of agglutination in which the agglutination of latex is made to carry on in a suitable vessel, the agglutinated clots are made separated from the non-agglutinated particles of latex by means of the centrifugal precipitation of the reactant liquid, and then the transmissibity of its supernatant liquid comes to be measured. In this case, however, the provision of a centrifugal separator and a relative operational process are required, all the more the measurement becoming complicated.
In addition to the above, there is still more known a method for detecting the presence of the agglutination through the measurement of the viscosity with regard to common latexes in the field other than the immunochemistry. This method also involves various problems such as the consumption of a comparatively great deal of samples, the intricacy in the temperature control and working curves, and so on, therefore being not able to expect the exactitude of the measurement.